Streptolysin S is required for Streptococcus pyogenes nasopharyngeal and skin infection in HLA-transgenic mice.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

Analysis of Toxicity of Recombinant Pneumolysin from Streptococcus pneumoniae.

We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.

Analysis of Immunobiological Properties of Recombinant Streptococcus pneumoniae Pneumolysin.

We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcus pneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

Construction of a pneumolysin deficient mutant in streptococcus pneumoniae serotype 1 strain 519/43 and phenotypic characterisation.

Streptococcus pneumoniae capsular serotype 1 continues to pose a huge infectious disease burden in low- and middle-income countries, particularly in West Africa. However, studies on this important serotype have been hampered by the inability to genetically modify these strains. In this study we have genetically modified a serotype 1 strain (519/43), the first time that this has been achieved for this serotype, providing the methodology for a deeper understanding of its biology and pathogenicity. As proof of principle we constructed a defined pneumolysin mutant and showed that it lost its ability to lyse red blood cells. We also showed that when mice were infected intranasally with the mutant 519/43Δply there was no significant difference between the load of bacteria in lungs and blood when compared to the wild type 519/43. When mice were infected intraperitoneally there were significantly fewer bacteria recovered from blood for the mutant 519/43Δply strain, although all mice still displayed signs of disease. Our study demonstrates S. pneumoniae serotype 1 strains can be genetically manipulated using our methodology and demonstrate that the ability to cause pneumonia in mice is independent of active pneumolysin for the 519/43 serotype 1 strain.

Streptococcal sagA activates a proinflammatory response in mast cells by a sublytic mechanism.

Mast cells are implicated in the innate proinflammatory immune defence against bacterial insult, but the mechanisms through which mast cells respond to bacterial encounter are poorly defined. Here, we addressed this issue and show that mast cells respond vividly to wild type Streptococcus equi by up-regulating a panel of proinflammatory genes and by secreting proinflammatory cytokines. However, this response was completely abrogated when the bacteria lacked expression of sagA, whereas the lack of a range of other potential virulence genes (seeH, seeI, seeL, seeM, hasA, seM, aroB, pyrC, and recA) had no effect on the amplitude of the mast cell responses. The sagA gene encodes streptolysin S, a lytic toxin, and we next showed that the wild type strain but not a sagA-deficient mutant induced lysis of mast cells. To investigate whether host cell membrane perturbation per se could play a role in the activation of the proinflammatory response, we evaluated the effects of detergent- and pneumolysin-dependent lysis on mast cells. Indeed, exposure of mast cells to sublytic concentrations of all these agents resulted in cytokine responses of similar amplitudes as those caused by wild type streptococci. This suggests that sublytic membrane perturbation is sufficient to trigger full-blown proinflammatory signalling in mast cells. Subsequent analysis showed that the p38 and Erk1/2 signalling pathways had central roles in the proinflammatory response of mast cells challenged by either sagA-expressing streptococci or detergent. Altogether, these findings suggest that sagA-dependent mast cell membrane perturbation is a mechanism capable of activating the innate immune response upon bacterial challenge.

Effects of Anaerobic Culturing on Pathogenicity and Virulence-Related Gene Expression in Pneumococcal Pneumonia.

BACKGROUND: The pathogenicity of Streptococcus pneumoniae under anaerobic conditions remains largely unknown. We examined the pathogenicity of S. pneumoniae cultured under anaerobic conditions in a murine model of pneumococcal pneumonia. METHODS: Mice were infected with S. pneumoniae grown under anaerobic or aerobic conditions. The pathogenic effects in vivo in the lower airway tract were then compared. The effect of anaerobic culture on lytA/ply transcript levels in vitro and in vivo were analyzed by quantitative real-time polymerase chain reaction. RESULTS: Mice inoculated with anaerobically cultured S. pneumoniae exhibited significantly lower survival rates and higher bacterial loads in the lungs and blood as compared to those infected with aerobically cultured S. pneumoniae. Aerobically cultured S. pneumoniae in the early log phase of growth was also able to induce severe pneumonia at levels equivalent to those of anaerobic S. pneumoniae. However, ply/gyrB transcript levels were significantly increased in the lungs of mice infected with anaerobically grown S. pneumoniae. In vitro, S. pneumoniae grown under anaerobic culture conditions demonstrated greater proliferation than S. pneumoniae grown under aerobic culture conditions, and bacterial concentrations were maintained for 24 hours without detectable upregulation of lytA messenger RNA. CONCLUSIONS: S. pneumoniae grown under anaerobic conditions had the potential to induce severe invasive bacteremic pneumococcal pneumonia in a manner different from that of S. pneumoniae grown under aerobic conditions.

Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

A Cross-Reactive Protein Vaccine Combined with PCV-13 Prevents Streptococcus pneumoniae- and Haemophilus influenzae-Mediated Acute Otitis Media.

Acute otitis media is one of the most common childhood infections worldwide. Currently licensed vaccines against the common otopathogen Streptococcus pneumoniae target the bacterial capsular polysaccharide and confer no protection against nonencapsulated strains or capsular types outside vaccine coverage. Mucosal infections such as acute otitis media remain prevalent, even those caused by vaccine-covered serotypes. Here, we report that a protein-based vaccine, a fusion construct of epitopes of CbpA to pneumolysin toxoid, confers effective protection against pneumococcal acute otitis media for non-PCV-13 serotypes and enhances protection for PCV-13 serotypes when coadministered with PCV-13. Having cross-reactive epitopes, the fusion protein also induces potent antibody responses against nontypeable Haemophilus influenzae and S. pneumoniae, engendering protection against acute otitis media caused by emerging unencapsulated otopathogens. These data suggest that augmenting capsule-based vaccination with conserved, cross-reactive protein-based vaccines broadens and enhances protection against acute otitis media.

Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae.

The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2-/- mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2-/- mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4+ T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2-/- DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

Epigallocatechin gallate inhibits Streptococcus pneumoniae virulence by simultaneously targeting pneumolysin and sortase A.

Streptococcus pneumoniae (pneumococcus), the causative agent of several human diseases, possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. Pneumolysin (PLY), an important virulence factor, is a member of the cholesterol-dependent cytolysin family and has cytolytic activity. Sortase A (SrtA), another crucial pneumococcal virulence determinate, contributes greatly to the anchoring of many virulence-associated surface proteins to the cell wall. In this study, epigallocatechin gallate (EGCG), a natural compound with little known antipneumococcal activity, was shown to directly inhibit PLY-mediated haemolysis and cytolysis by blocking the oligomerization of PLY and simultaneously reduce the peptidase activity of SrtA. The biofilm formation, production of neuraminidase A (NanA, the pneumococcal surface protein anchored by SrtA), and bacterial adhesion to human epithelial cells (Hep2) were inhibited effectively when S. pneumoniae D39 was cocultured with EGCG. The results from molecular dynamics simulations and mutational analysis confirmed the interaction of EGCG with PLY and SrtA, and EGCG binds to Glu277, Tyr358, and Arg359 in PLY and Thr169, Lys171, and Phe239 in SrtA. In vivo studies further demonstrated that EGCG protected mice against S. pneumoniae pneumonia. Our results imply that EGCG is an effective inhibitor of both PLY and SrtA and that an antivirulence strategy that directly targets PLY and SrtA using EGCG is a promising therapeutic option for S. pneumoniae pneumonia.

Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice.

Hypervirulent group A streptococcus (GAS) can inhibit neutrophil recruitment and cause systemic infection in a mouse model of skin infection. The purpose of this study was to determine whether platelet-activating factor acetylhydrolase Sse and streptolysin S (SLS) have synergistic contributions to inhibition of neutrophil recruitment and systemic infection in subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. Deletion of sse and sagA in MGAS315 synergistically reduced the skin lesion size and GAS burden in the liver and spleen. However, the mutants were persistent at skin sites and had similar growth factors in nonimmune blood. Thus, the low numbers of Δsse ΔsagA mutants in the liver and spleen were likely due to their reduction in the systemic dissemination. Few intact and necrotic neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and necrotic cells were present at the edge of Δsse mutant infection sites on day 1 and at the edge of and inside Δsse mutant infection sites on day 2. ΔsagA mutant infection sites had massive numbers of and few intact neutrophils at the edge and center of the infection sites, respectively, on day 1 and were full of intact neutrophils or necrotic cells on day 2. Δsse ΔsagA mutant infection sites had massive numbers of intact neutrophils throughout the whole infection site. These sse and sagA deletion-caused changes in the histological pattern at skin infection sites could be complemented. Thus, the sse and sagA deletions synergistically enhance neutrophil recruitment. These findings indicate that both Sse and SLS are required but that neither is sufficient for inhibition of neutrophil recruitment and systemic infection by hypervirulent GAS.

Anticytotoxin Effects of Amentoflavone to Pneumolysin.

Pneumolysin (PLY) is a devastating bacterial protein toxin of Streptococcus pneumoniae that punctures the cytomembrane, leading to pathological reactions, such as cell disruption and inflammation. Drugs capable of closely impacting the toxin are considered advantageous in the treatment of bacterial infections. Amentoflavone (AMF) is a chemical substance extracted from traditional Chinese herbs. Previous studies have demonstrated that AMF has multiple pharmacological effects and mentioned without attenuating pneumolysin-mediated cytotoxicity. This work focuses on the influence of AMF on inhibitory hemolytic mechanisms. AMF interacts with the toxin at Ser254, Glu277, Arg359, and effectively weakens the oligomerization of wild-type PLY and provides considerable protection against pneumolysin-mediated human alveolar epithelial (A549) cell damage. The results of our study demonstrate that AMF could be a candidate against pneumolysin-related injury.

[Research progress in pneumolysin].

Pneumolysin is a multifunctional virulence factor expressed by Streptococcus pneumoniae. Pneumolysin includes 4 domains and is a member of cholesterol-dependent cytolysins. Pneumolysin has extensive cytotoxicity to a range of host cells. Furthermore, pneumolysin can activate complement classical pathway, and induce macrophages and monocytes to produce proinflammatory cytokines, mediate host immune responses. Consequently, pneumolysin is a potential candidate target for research and development of vaccines and drugs. In this review, the latest research progresses on the structure and function of pneumolysin, and related vaccines are discussed.

Structure-based functional studies for the cellular recognition and cytolytic mechanism of pneumolysin from Streptococcus pneumoniae.

Cholesterol-dependent cytolysins (CDCs) contribute to various pathogenesis by Gram-positive bacterial pathogens. Among them, pneumolysin (PLY) produced by Streptococcus pneumoniae is a major contributor to pneumococcal infections. Despite numerous studies of the cytolytic mechanism of PLY, little structural information on its interactions with a specific receptor of the cell membrane is available. We report here the first crystal structures of PLY in an apo-form and in a ternary complex with two mannoses at 2.8Å and 2.5Å resolutions, respectively. Both structures contained one monomer in an asymmetric unit and were comprised of four discontinuous domains, similar to CDC structures reported previously. The ternary complex structure showed that loop 3 and the undecapeptide region in domain 4 might contribute to cellular recognition by binding to mannose, as a component of a specific cell-surface receptor. Moreover, mutational studies and docking simulations for four residues (Leu431, Trp433, Thr459, and Leu460) in domain 4 indicated that Leu431 and Trp433 in the undecapeptide might be involved in the binding of cholesterol, together with the Thr459-Leu460 pair in loop 1. Our results provide structure-based molecular insights into the interaction of PLY with the target cell membrane, including the binding of mannose and cholesterol.